First Report of Fusarium kyushuense Causing Leaf Spot on Sweet Persimmon in China.

Sweet persimmon (Diospyros kaki L.) is a fruit of significant nutritional and commercial value in Asia. In summer 2023, leaf spots were observed affecting 20 to 30% of sweet persimmon trees in a commercial orchard located in Gongcheng City, Guangxi, China. Initially, the infected leaves exhibited sparse light brown spots on their upper surface, which subsequently evolved into brown circular to irregular lesions encircled by a yellow halo. Eventually, these lesions became densely distributed across the leaves leading to insufficient nutrient accumulation in the fruit. To isolate the pathogen, diseased leaves were cut into small pieces (5×5 mm), disinfected with 75% ethanol for 15 seconds, followed by 1% NaClO for 1minute, rinsed three times with sterile water, and then transferred onto potato dextrose agar (PDA) plates. The plates were then incubated in darkness for 3 days at 25°C. Pure cultures were obtained using the hyphal-tip method and single-spore isolation. On PDA, the colonies initially appeared fluffy and white after 24 hours, turning yellowish or red after 3 days. Macroconidia (average length of 26.1 µm in length × 4.3 µm in width, n = 50) exhibited dorsiventral curvature and were hyaline, with 3 to 5 septa. Microconidia (average length of 9.45 µm in length × 3.4 µm in width, n = 50) were hyaline, aseptate, and oval. Two representative isolates, Gxfky1 and Gxfky2, were selected for further molecular analyses. Their internal transcribed spacer (ITS) region rDNA gene were amplified via PCR and sanger sequenced (GenBank Accession Nos. PP506475, PP506593) using the primer pair ITS1/ITS4 (White et al. 1990), showing more than 99% sequence identity with Fusarium kyushuense type-material strain NRRL3509 (NR_152943) according to BLASTn analysis in NCBI. To further confirm the identity of the isolates, four gene sequences were amplified: RPB1 (PP532864, PP532865), RPB2 (PP532866, PP532867), TEF1 (PP580505, PP580506), and TUB2 (PP532862, PP532863), using the F5/G2R, 5f2/11ar, EF1/EF2, and T1/T2 primer sets, respectively (O'Donnell et al., 1997; O'Donnell et al., 2010). A multi-locus maximum likelihood phylogenetic analysis revealed that Gxfky1 and Gxfky2 clustered with strains F. kyushuense with 100% bootstrap support. Pathogenicity tests using Gxfky1 and Gxfky2 were conducted on leaves of two-year-old sweet persimmon plants using non-wound inoculation. Specifically, 5-mm mycelial plugs and sterile agar plugs were placed on six leaves and secured with cling film, with six plugs each for the inoculation treatment and negative control, respectively. They were then incubated in a greenhouse at room temperature (25 ± 2°C) with a relative humidity of 70 to 80%. After 5 days, the same symptoms on naturally infected plants were observed on leaves inoculated with mycelium, while no symptoms were observed on the controls. The same fungus were reisolated from the inoculated leaves and identified based on morphology and the TEF1 gene sequence, thus fulfilling Koch's postulates. Fusarium kyushuense has previously been reported to cause diseases in various plant species, including maize (Cao et al., 2021), rice (Wang et al., 2024), and tobacco (Wang et al., 2013). To our knowledge, this is the first report of F. kyushuense causing leaf spot on sweet persimmon in China, which expands the known host range of this pathogen.

First report of Fusarium falciforme causing root rot of pomegranate (Punica granatum L.) in China.

Pomegranate (Punica granatum L.) is a deciduous shrub or small tree that is native to Iran and Afghanistan. It is also a commercially important fruit tree in China and worldwide. In the summer of 2022, a serious root rot disease occurred in some pomegranate orchards in Xichuan County(32º42´ N, 111º48´ E), Henan Province, China, with an incidence of ~30%. Symptoms included leaf yellowing and wilting, root browning and rotting, and stem-base cracking, eventually leading to defoliation and death. To isolate the causal agent, small pieces (5×5 mm) of diseased root from six trees were surface-sterilized by dipping in 2% NaClO for 8 min followed by 70% ethanol for 15 s, rinsed five times with sterile water, and plated on potato dextrose agar (PDA), then incubated at 28°C in the dark for 5 days. Fifteen pure fungal isolates with the same morphological characteristics were obtained from 24 pieces of roots. All isolates produced white fluffy mycelia. Microconidia were hyaline, oval or reniform, with zero to one septa and dimensions of 7.1 to 19.9 (average 14.5 )× 3.8 to 8.0 (average 5.6) µm (n = 100). Macroconidia were sickle-shaped, one to four septate, and 20.1 to 40.8 (average 26.5) × 4.8 to 8.6 (average 6.5) µm (n = 100). Chlamydospores were spherical, single, in pairs or chains, and 5.6 to 9.8 (average 6.8) µm in diameter (n = 100). Based on the above characteristics, the pathogens were identified as Fusarium sp. (Leslie and Summerell 2006). Genomic DNA was extracted from mycelia of two representative isolates Fs1 and Fs3. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and RNA polymerase II second largest subunit (RPB2) sequences were PCR amplified using primer pairs of ITS1/ITS4, EF1/EF2, and RPB2-5f2/RPB2-7cr, RPB2-7cf/RPB2-11ar (O'Donnell et al., 2022), respectively. BLAST analysis showed that the ITS, TEF-1α and RPB2 sequences of isolates Fs1(GenBank accession nos. OK001765, OQ921726 and OQ928396) and Fs3 (GenBank accession nos. OK001771, OQ921727 and OQ928397) showed 99%-100% identity with multiple GenBank sequences of Fusarium falciforme (KY617066, MN064683, KF255514, OQ933361, KY556711 and ON331935). A phylogenetic tree based on concatenated sequences of ITS, TEF-1α and RPB2 using maximum-likelihood analysis revealed that both isolates Fs1 and Fs3 were in the same clade with F. falciforme strains. Based on the morphological and molecular characteristics, the isolates were identified as members of F. falciforme. For pathogenicity testing, conidial suspensions (1×108 spores /mL) of isolates Fs1 and Fs3 were poured onto the roots of healthy pomegranate that had been planted in pots two months previously. Ten plants were inoculated for each isolate. Control plants were drenched with sterile water. After 3 months, inoculated plants developed leaf yellowing and wilting accompanied by root browning and rotting, much like symptoms observed in field plants. The same fungi re-isolated from the experimental plants were confirmed to be F. falciforme by morphology and sequence analysis. This is the first report of F. falciforme causing root rot on pomegranate. F. falciforme is a ubiquitous soil-borne pathogen that causes root rot on multiple plants around the world (Xu F., et al. 2022; Qiu R., et al. 2023). The results of pathogen identification are essential precursors to development of effective control of the disease.

[Observation on effect of five evolutive phases and six climatic factors on pregnancy and sterility].

Objective： To verify statements about the effect of five evolutive phases and six climatic factors in 《huang di nei jing》 on pregnancy and sterility. Methods： Data of missed abortion from Nanjing Maternity and Child Health Care Hospital between 2006-2020 and Yangzhou Maternity and Child Health Care Hospital between 2012-2020 were collected. According to Chinese sexagenary cycle heavenly stems and earthly branches of each year between 2006-2020 was determined. And then based on the heavenly stems and earthly branches, evolutive phases and six climatic factors of each year were established. After that data of missed abortion in each year was compared. According to relative statements in 《huang di nei jing》 , it was observed whether the number of missed abortion cases in someone year increased significantly. Results: Totally there were 28059 cases of missed abortion from the two hospital were collected between 2006-2020 and 2012-2020 respectively. It was found that in the year of Shaoyang phase fire sitian and Jueyin wind wood in the spring (that is the year of 2010 and 2016) the number of missed abortion cases was higher obviously than the other year. When the year of Taiyang cold water in the spring (that is the year of 2009 and 2015), there was a relatively increased number of missed abortion cases. Conclusions: Five evolutive phases and six climatic factors have impacts on human pregnancy and sterility. It was an adverse condition for human reproduction in the year of Shaoyang phase fire sitian and Jueyin wind wood in the spring as well as Taiyang cold water in the spring. Until to now we can still verify statements about the effect of five evolutive phases and six climatic factors on pregnancy and sterility in 《huang di nei jing》.

[Observation of effect of Wu Yun Liu Qi on reproductive hormones in varicocele patients].

Objective： To observe roproductive hormone levels in varicocele patients during a cycle (6 years) of Wu Yun Liu Qi, and explore whether the cycle had effect on the roproductive hormone levels. Methods： Data of roproductive hormone levels in varicocele patients from 2015 to 2020 were analyzed retrospectively. FSH、LH、T、PRL、E2 levels and T/E2 ratio were compared among the six years. According to Chinese sexagenary cycle heavenly stems and earthly branches of each year from 2015 to 2020 its yunqi characteristics were determined. Results: Totally data of 848 cases of varicocele patients were collected from 2015 to 2020. Among which, in 2015 (Yiwei year) there were 57 cases, in 2016 (Bingshen year) 83 cases, in 2017 (Dingyou year ) 133 cases, in 2018(Wuxu year) 156 cases, in 2019(Sihai year) 274 cases, and in 2020(Gengzi year) 145 cases. The levels of FSH、LH、PRL、T were not diferrent statistically from the six years except individual year. However, the level of E2 in 2016 when the Yunqi was Shao Yang Xiang Huo Si Tian and Jue Yin Feng Mu Zai Quan obviously higher than other years excpet 2018( All P< 0.05). And T/E2 ratio was lower in 2016 than other years except 2018 and 2020( All P< 0.05). Conclusions: Shi Xiang factors of Wu Yun Liu Qi had effect on roproductive hormone levels in varicocele patients, showing by higher E2 level in Yinshen year when the Shi Xiang factors may have bad effect on human fertility.

Eupalinolide B inhibits hepatic carcinoma by inducing ferroptosis and ROS-ER-JNK pathway.

Primary hepatic carcinoma is a common malignant tumor. The classic molecular targeted drug sorafenib is costly and is only effective for some patients. Therefore, it is of great clinical significance to search for new molecular targeted drugs. Eupalinolide B (EB) from Eupatorium lindleyanum DC. is used to treat chronic tracheitis in clinical practice. However, the role of EB in hepatic carcinoma is unknown. In this study, we first measure the effect of EB on tumor growth in a xenograft model and PDX model. The cell proliferation and migration are also detected in human hepatocarcinoma cell lines (SMMC-7721 and HCCLM3). Then, we investigate cell cycle, cell apoptosis, cell necrosis, cell autophagy, and ferroptosis by flow cytometry, western blot analysis and electron microscopy. The results demonstrate that EB exerts anti-proliferative activity in hepatic carcinoma by blocking cell cycle arrest at S phase and inducing ferroptosis mediated by endoplasmic reticulum (ER) stress, as well as HO-1 activation. When HO-1 is inhibited, EB-induced cell death and ER protein expression are rescued. The migration-related mechanism consists of activation of the ROS-ER-JNK signaling pathway and is not connected to ferroptosis. In summary, we first discover that EB inhibits cell proliferation and migration in hepatic carcinoma, and thus EB is a promising anti-tumor compound that can be used for hepatic carcinoma.

First Report of Neofusicoccum parvum Causing Leaf Spot on Geodorum eulophioides in China.

Geodorum eulophioides Schltr. is a critically endangered orchid listed in the International Union for Conservation of Nature (IUCN) Red List of threatened species. At present, only two natural populations were found in China. It has important scientific and ornamental values because of its uniqueness. During the summer of 2019, a black leaf spot disease occurred on G. eulophioides, in Yachang Orchid National Nature Reserve (E106°13'32″，N24°44'19″) in Guangxi province, China. More than 60% of leaves of these plants were infected. The disease symptoms initially appeared as small yellow circular spots, which enlarged into irregular brown spots (6 to 9 cm length and 3 to 5 cm width). In later stages of the disease development, the center of the spots became dark brown with a clear edge and surrounded by a yellow halo. In severe infections, the spots coalesced covering the entire leaf. Six symptomatic leaves were collected from three infected plants, surface sterilized in 75% ethanol for 15 s and 0.1% HgCl2 for 4 min, and subsequently washed three times with sterile water, then plated onto potato dextrose agar (PDA), and incubated at 28℃ for three days. Eighteen fungal cultures with similar morphological characteristics were obtained from the infected tissues. Colonies were initially white, then turned dark grey after nine days. To induce sporulation, isolates were grown on 2% water agar and incubated under UVA light at 28℃ for nine days. Three isolates were selected for morphological characterization. Conidia were hyaline, unicellular, nonseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and ranged from 10.7 to 16.6 µm (avg. 13.8 µm) × 4.1 to 6.7 µm (avg. 5.1 µm) (n=50). The isolate DBL-1 was selected as a representative for molecular identification. Genomic DNA was extracted and used for PCR to amplify the rDNA internal transcribed spacer region (ITS), translation elongation factor 1-alpha gene (EF1-α), and beta-tubulin gene (TUB2), using the primer pairs ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R（Alves et al. 2008；Carbone & Kohn, 1999）, and T1/T2 (O'Donnell et al., 1997), respectively. The obtained ITS sequence (GenBank Accession No. MN918440), EF1-α sequence (MN963815), and TUB2 sequence (MN963816) showed >99% homology with several GenBank sequences of Neofusicoccum parvum (JX513636, KU997497 for ITS, KU997261, MH252401 for EF1-α, and KJ841779, MK412882 for TUB2, respectively). Based on morphological characteristics of the asexual morph and maximum likelihood analyses of a combined rDNA-ITS, EF1-α and TUB2 gene sequences, was identified as N. parvum. Pathogenicity test was performed using isolate DBL-1 by inoculating 3 leaves of G. eulophioides plants. The test was repeated three times. Each leaf was wounded using a sterile needle, and a mycelial plug (6 mm diameter) harvested from the periphery of a 3-day-old colony grown on PDA was placed on each wound. Plants were then covered with plastic bags to maintain high relative humidity of 90% and kept at 28℃ in a greenhouse under natural daylight conditions. An equal number of leaves on the same plant were inoculated using sterile PDA plugs and served as mock inoculated controls. After three days, all the inoculated leaves showed black spot symptoms resembling those observed in the field, whereas controls remained symptomless. The fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. N. parvum has been reported to cause leaf spot disease on Myristica fragrans (Jayakumar, et al., 2011), Ginkgo biloba (Mirhosseini, et al., 2014), Vitis heyneana (Wu, et al., 2015), and Hevea brasiliensis (Liu et al., 2017), respectively. To the best of our knowledge, this is the first report of N. parvum causing leaf spot disease on G. eulophioides in China. The disease control measures and in-situ conservation method need to be strengthened to protect this rare species.

[Differential expression profiles of miRNAs in the vaginal tissue of the women with lubrication disorders].

OBJECTIVE: To explore the pathogenesis of lubrication disorder (LD), a most common type of female sexual dysfunction affecting women's physical health and conception, and find the therapeutic targets for its treatment and prevention. METHODS: We chose 3 LD patients and 3 healthy controls in Nanjing Maternal and Child Health Hospital, extracted their vaginal epithelial RNA for high-throughput miRNA sequencing, screened differentially expressed miRNAs for hierarchical cluster analysis, target gene prediction and gene ontology (GO) and KEGG pathway enrichment analyses. Finally, we verified the sequencing results by real-time fluorescent quantitative PCR. RESULTS: Totally 1 673 miRNAs were predicted by high-throughput sequencing and 64 likely to be the targets for the treatment of LD were screened, including 25 up-regulated more than 4 times and 39 down-regulated more than 4 times in the LD patients compared with the healthy controls. The neuron projection morphogenesis and AMPK signaling pathway were the most significant enrichment GO term and KEGG pathway. CONCLUSIONS: miRNAs are expressed differentially in LD patients. These miRNAs and target genes may be related to the occurrence of LD, and those that are expected to be the targets for the treatment of LD have important theoretical significance and potential application value.

[Effect of bilateral microsurgical subinguinal varicocelectomy on asthenozoospermia and oligozoospermia: A report of 109 cases].

OBJECTIVE: To investigate the effects of bilateral microsurgical subinguinal varicocelectomy (BMSV) in patients with asthenozoospermia or oligozoospermia. METHODS: Totally 147 patients with male infertility received BMSV from January 2018 to May 2019, of whom 109 had complete data recorded. We retrospectively analyzed the clinical data, including the total sperm count per ejeculate, sperm concentration and sperm motility before and after surgery, and the rate of natural conception achieved during the follow-up. RESULTS: BMSV achieved a total effectiveness rate of 79.00% in improving the percentage of progressively motile sperm (a rise of ≥20%) and a marked effectiveness rate of 70.00% (a rise of ≥50%) in the 100 cases of asthenozoospermia as compared with the baseline, with a mean recovery time of (110.13 ± 37.43) days. Besides, a total effectiveness rate of 74.29% (an increase of ≥20%) and a marked effectiveness rate of 71.43% (an increase of ≥50%) were attained in the improvement of sperm concentration in the 35 cases of oligozoospermia, with a mean recovery time of (117.00 ± 48.79 ) days. A natural conception rate of 30.30% was observed during the follow-up. No severe adverse events occurred postoperatively. CONCLUSIONS: BMSV is significantly effective for the treatment of asthenozoospermia and oligozoospermia.

[HPV infection of the external genitalia in men whose female partners have cervical HPV infection].

OBJECTIVE: Human papilloma virus (HPV) is a necessary cause of cervical cancer and is also closely related to penile cancer, oropharyngeal cancer, and anal cancer in males. However, few studies are reported on male HPV. This study aimed to investigate HPV infection of the external genitalia in men whose female partners have cervical HPV infection. METHODS: We collected the relevant data on the male outpatients whose partners had cervical HPV infection in our Department of Urology and Andrology from August to December 2016. We obtained samples with nylon swabs from the glans penis, corona, inner layer of the prepuce and penile body and detected different types of HPV infection using the Hybribio HPV typing kit, PCR and membrane hybridization. RESULTS: Valid data were collected from 140 males, which showed 83.5% of HPV infection of the external genitalia, including 60 cases of HPV6 (43.2%), 27 cases of HPV16 (19.4%), 14 cases of HPV39 (10.1%), 13 cases of HPV18 (9.4%), 13 cases of HPV58 (9.4%), and 13 cases of HPV52 (9.4%). Redundant prepuce was found in 75.5% of the males, but there was no statistically significant difference in the incidence rate of HPV infection between the normal and redundant prepuce groups (P > 0.05). CONCLUSIONS: Men who have the female partners with positive cervical HPV are at high risk of HPV infection and therefore need to be screened and treated so as to reduce HPV infection in both sexes.

[Relationship of abnormal sperm DNA methylation with early spontaneous abortion].

OBJECTIVE: To investigate the relationship between the abnormal sperm DNA methylation level and early spontaneous abortion. METHODS: We randomly selected 98 males who met the inclusion criteria and whose wives suffered from unexplained abortion or embryo abortion, and included another 46 normal healthy men present for pre-pregnancy check-up as controls. We examined the semen quality and sperm morphology, obtained the sperm DNA fragmentation index (DFI) by modified sperm chromatin dispersion, and measured the sperm DNA methylation level using the methylated DNA quantification kit and the colorimetric method. RESULTS: Compared with the normal controls, the men in the unexplained abortion group showed a significantly lower rate of big-halo sperm (ï¼»45.50 ± 26.27ï¼½ vs ï¼»36.49 ± 23.06ï¼½%, P = 0.038), a higher rate of abnormal-head sperm (ï¼»77.08± 12.21ï¼½ vs ï¼»81.09± 10.89ï¼½%, P = 0.049), and a lower level of sperm DNA methylation (ï¼»0.47 ± 0.33ï¼½ vs ï¼»0.36 ± 0.26ï¼½ ng/µl, P = 0.035). The sperm DNA methylation level was positively correlated with the percentage of big-halo sperm (OR=0.546, P<0.01). Multivariate regression analysis manifested that sperm head abnormality was an independent risk factor of early spontaneous abortion or embryo abortion (OR=1.032, P = 0.049), while the high methylation level was protective factor against early spontaneous abortion or embryo abortion (OR=0.244, P = 0.03). CONCLUSIONS: The abnormal level of sperm DNA methylation may be one of the important reasons for early spontaneous abortion or embryo abortion.

[Semen quality and its influencing factors in preconception males in Nanjing area].

OBJECTIVE: To investigate the semen quality and its influencing factors in preconception males in Nanjing area so as to provide some evidence for working out effective intervention measures. METHODS: Totally 687 men receiving preconceptional physical examination were enrolled in this study. A questionnaire survey was conducted among the subjects along with an analysis of their semen quality. RESULTS: The median of sperm concentration was 63.3 x 10(6)/ml (95% CI [19.88-119] x 10(6)/ml). The median of grade a sperm was 33.03% (95% CI [19.38-55.05]%), that of grade a + b sperm was 52.08% (95% CI [39.53-69.37]%), and that of teratosperm was 91.75% (95% CI [69-100]%). The median concentration of seminal plasma PMN-elastase was 195.55 ng/ml (95% CI [76.16-3330.38] ng/ml) and that of seminal plasma zinc was 7.62 µmol/L (95% CI [1.5-23, 45] µmol/L). The positive rates of Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), and Gardnerella vaginalis (GV) were 42.4%, 0.3%, and 2.4%, respectively. The median of sperm DNA fragmentation index (DFI) of those whose wives had a history of adverse pregnancy was 20.25% (95% CI [2.15-68.25]%). Multivariate logistic regression analysis suggested that mental stress (OR 1.567, 95% CI [1.081-2.27]) and sedentariness (OR 1.772, 95% CI [1.211-2.592]) were independent risk factors for asthenospermia. CONCLUSION: The sperm quality of preconception males in Nanjing area is not encouraging, and it can be improved by changing undesirable lifestyle and reducing mental stress.

[Relationship between epigenetics of sperm and embryogenesis].

Epigenetics comprises the modifications made in gene expressions without changing the DNA sequence itself. Significant epigenetic changes take place during spermatogenesis and fertilization and exert direct influences on embryogenesis. This article provides an overview of the latest researches on epigenetics of male germ cells and a brief discussion on the correlation of sperm with embryogenesis in four aspects: DNA methylation, histone modification, regulation of non-coding RNAs, and genomic imprinting.

[Protective effect of pAdxsi-ERbeta adenovirus transfection on penile vascular endothelium in ERbetaKO mice].

OBJECTIVE: To investigate the effect of the overexpression of the ERbeta gene on the penile vascular endothelium of ERbeta knockout (ERbetaKO) mice and its molecular mechanisms. METHODS: We randomly divided 12 ERbetaKO male mice into groups A (ERbetaKO + TNFalpha + pAdxsi-ERbeta) and B (ERbetaKO + TNFalpha + empty virus), the former treated by pAdxsi-ERbeta adenovirus transfection, the latter with empty virus, and meanwhile both injected intraperitoneally with TNFalpha at 6 microg per kg body weight per d for 14 days. Then we observed the erectile function of the mice by APO, determined the changes of the endothelial markers CD34 and vWF by immunohistochemical staining, and detected the expressions of the relevant molecules in the eNOS-NO pathway by RT-PCR, Western blot and immunohistochemistry. RESULTS: Compared with group B, group A showed a significantly increased number of penile erections (0.50 +/- 0.55 vs 2.17 +/- 0.41, P < 0.05), shortened erectile latency ([28.83 +/- 1.33] min vs [24.00 +/- 1.27] min, P < 0.05), enriched CD34 and vWF markers (0.67 +/- 0.52 vs 1.50 +/- 0.55 and 0.50 +/- 0.55 vs 1.33 +/- 0.52, both P < 0.05), elevated expressions of eNOS and Cam (RT-PCR: 1.38 +/- 0.03 vs 1.62 +/- 0.05 and 1.02 +/- 0.09 vs 1.42 +/- 0.05, both P < 0.05; Western blot: 1.27 +/- 0.04 vs 1.55 +/- 0.07 and 0.76 +/- 0.05 vs 0.95 +/- 0.08, both P < 0.05), and reduced expression of caveolin-1 (RT-PCR: 2.13 +/- 0.13 vs 1.72 +/- 0.08, P < 0.05; Western blot: 3.99 +/- 0.16 vs 3.40 +/- 0.14, P < 0.05). The results of RT-PCR were consistent with those of Western blot. CONCLUSION: The ERbeta gene protects the penile vascular endothelium via the eNOS-NO pathway.

[Seminal parameters at different times of reanalysis of the normal semen samples detected in initial examination and their correlation with sperm DNA damage].

OBJECTIVE: To investigate the differences in semen quality at different times of reanalysis and the correlation of sperm DNA fragmentation index (DFI) with sperm motility alteration using semen samples completely liquefied and normal in initial examination. METHODS: We analyzed 127 semen samples up to the inclusion criteria with the computer-assisted semen analysis (CASA) system at 15, 30 and 60 min after semen collection, and obtained sperm morphology parameters and DFI by Shorr staining and acridine orange test (AOT) , respectively. RESULTS: Sperm concentration, and the percentages of grades a and b sperm showed no statistically significant differences at the three time points (P > 0.05). The percentages of grades a + b and a + b + c sperm were significantly higher at 15 min than at 30 and 60 min after semen collection (P < 0.05), but with no significant difference between the latter two time points (P > 0.05). The incidence of alternation from normal to abnormal in at least one index of sperm motility at different times was 25.2%, but there were no significant differences in sperm DFI and morphology between the normal and abnormal groups (P > 0.05). Among the altered parameters of sperm motility from 15 to 60 min, the percentages of grades a, a + b and a + b + c sperm were all positively correlated with sperm DFI (P < 0.05). CONCLUSION: Semen samples completely liquefied within 15 min after collection and normal in initial examination, when reanalyzed at 30 and 60 min, showed significant decreases in the percentages of grades a + b and a + b + c sperm, but not in the percentages of grades a and b sperm, and the parameters of sperm motility might be abnormal. Thus, at least 2 sperm analyses are required for a comprehensive evaluation of fertility. Significant difference between the results of the two analyses, and particularly a markedly reduced percentage of rapidly progressive sperm, might indicate sperm DNA damage, and thus the necessity of sperm DNA damage detection.

Aptamer-Based Functionalized SERS Biosensor for Rapid and Ultrasensitive Detection of Gastric Cancer-Related Biomarkers.

Background: Gastric cancer (GC) as is the second deadliest malignancy still lacks rapid, simple and economical detection and early clinical screening techniques. Surface-enhanced Raman spectroscopy (SERS) is a spectroscopic technique based on the surface plasmon resonance of precious metal nanoparticles, which can effectively detect low-abundance tumor markers. Combining SERS technology with sensors has high potential in the diagnosis and screening of GC. Methods: A novel Au/Si nano-umbrella array (Au/SiNUA) was prepared as a SERS substrate and the substrate was functionalized using the corresponding tumor marker aptamers for the detection of clinical biological samples using a one-step recognition release mechanism. Optimization of aptamer and complementary chain concentrations and detection time for optimal sensor preparation. Results: Au/SiNUA were tested to have good SERS enhancement activity. The proposed aptamer biosensor has good specificity and stability, with a low detection time of 18 min and a limit of detection (LOD) at the fM level, which is superior to most of the methods reported so far; and the accuracy of the clinical assay is comparable to that of the ELISA method. The expression levels of PDGF-B and thrombin in the serum of GC patients and healthy individuals can be effectively detected and differentiated. Conclusion: The ultrasensitive and specific aptamer biosensor is highly feasible for the diagnosis and screening of GC and has good application prospects.

[Human papillomavirus infection correlates with redundant prepuce or phimosis in the patients'sexual partners in Nanjing urban area].

OBJECTIVE: To investigate the incidence of human papillomavirus (HPV) infection in women and its correlation with redundant prepuce or phimosis in the patients' sexual partners. METHODS: We conducted a questionnaire investigation among the women outpatients at the cervical disease clinic of Nanjing Maternity and Child Health Hospital from May to December 2011. We obtained information on their sexual life and determined whether their sexual partners had redundant prepuce or phimosis according to the schematic illustrations we offered. We used biology-hybridize HPV-type test kit, PCR and hybrid membrane methods for detection of different HPV genotypes in cervical exfoliated cells, taking any type of HPV detected as positive. We made between-group comparisons by chi-square test and analyzed independent risk factors by non-conditional logistic regression analysis. RESULTS: Of the 2 040 questionnaires, 1 568 were collected and 1 110 (71%) accepted as valid by inclusion criteria. Among the 1 110 subjects, 566 (50.9%) were infected with HPV, and 445 (78.6%) of the infected women admitted that their sexual partners had redundant prepuce or phimosis. The most frequent infection type was HPV16 (34.2%), followed by HPV58 (28.1%), HPV52 (20.2%) and HPV18 (10.8%). Multivariate logistic regression analysis suggested that redundant prepuce or phimosis in the women's sexual partners was an independent risk factor for HPV infection (OR 3.387, 95%CI [2.491-4.607]). CONCLUSION: In Nanjing urban area, the majority of the sexual partners of the HPV-infected women have redundant prepuce or phimosis, which is an independent risk factor for female cervical HPV infection. Male circumcision is necessitated in Nanjing to reduce the incidence of cervical HPV infection.

[Treatment of varicocele by microsurgical varicocelectomy: report of 94 cases].

OBJECTIVE: To investigate the clinical effect of microsurgical varicocelectomy in the treatment of varicocele. METHODS: We retrospectively analyzed the clinical data of 94 varicocele patients treated by microsurgical varicocelectomy, of whom 36 complained of testicular pain or dragging and distending discomfort, and 58 infertility or oligoasthenospermia. Microsurgical varicocelectomy was performed through inguinal or subinguinal approach. Complications were observed and semen parameters detected through follow-up visits at 1, 3 and 6 months after the operation. RESULTS: Testicular pain or discomfort disappeared in 21 (65.6%) of the 32 followed-up patients who had complained of such symptoms, was relieved in 6 (18.8%), and remained unimproved in the other 5 (15.6%). The 56 followed-up patients with infertility or oligoasthenospermia all showed significantly improved sperm concentration and motility (grade a + b sperm), (15.47 +/- 3.21) x 10(6)/ml and (13.34 +/- 5.16)% at 3 months, and (18.39 +/- 4.05) x 10(6)/ml and (17.23 +/- 4.69)% at 6 months after operation, as compared with (8.26 +/- 1.68) x 10(6)/ml and (5.25 +/- 1.09)% preoperatively (P < 0.01). CONCLUSION: Microsurgical varicocelectomy can effectively improve symptoms and semen parameters in varicocele patients.

[Protective effect of ERbeta on penile vascular endothelium in mice].

OBJECTIVE: To investigate the protective effect of ERbeta on the penile vascular endothelium in mice. METHODS: We randomly selected 12 ERbeta knockout (ERbetaKO) and 12 C57BL/6 male mice, and divided them into four groups: normal control, ERbetaKO, ERbetaKO + TNFalpha, and wild-type + TNFalpha group. The former two were treated with normal saline, while the latter two by intraperitoneal injection of TNFalpha at 6 microg/(kg x d) for 14 days. Then we observed the spontaneous erectile response induced by APO and changes of the endothelial cells by immunohistochemical staining of CD34 and vWF, and detected cell apoptosis in the penile cavernous tissue by TUNEL. RESULTS: Compared with the normal control group, the ERbetaKO group showed significantly increased erectile latency (P<0.05), but no significant difference in the number of erections; the ERbetaKO + TNFalpha and wild-type + TNFalpha groups, too, exhibited remarkably longer erectile latency (P<0.05) but fewer erections (P<0.05), with even more obvious changes in the ERbetaKO + TNFalpha group. The expressions of CD34 and vWF were significantly reduced in the ERbetaKO group (2.25 +/- 0.50 and 2.00 +/- 0.00), ERbetaKO + TNFalpha group (0.25 +/- 0.50 and 0.33 +/- 0.58) and wild-type + TNFalpha group (1.50 +/- 0.58 and 1.25 +/- 0.50) as compared with those in the control (3.00 +/- 0.00 and 2.75 +/- 0.50) (P<0.05), even lower in the ERbetaKO + TNFalpha than in the wild-type + TNFalpha group (P<0.05). Apoptotic cells were found only in the ERbetaKO + TNFalpha group. CONCLUSION: After ERbeta knockout and especially after treated with the endothelial injury factor TNFalpha, endothelial cells are decreased in the penile vessels in mice, which suggests that ERbeta has a protective effect on the penile cavernous sinus endothelium.

[Estimation of PMI using late postmortem phenomena in the basis of 49 cases].

OBJECTIVE: To discuss the influencing factors of using late postmortem phenomena to estimate PMI and to provide experience for an accurate estimation. METHODS: Forty-nine corpses of late postmortem were collected in Shaoxing City, Zhuji area from 2004 to 2011. The related factors were analyzed including season, scene, estimated PMI, exact PMI, cause of death and main factors effected PMI, etc. RESULTS: Of all 49 cases, 20 corpses were outdoor, 11 were indoor and 18 were in water. Thirty-seven cases were successful to estimate PMI and 12 cases were unsuccessful. The main factors affected PMI were infection, poisoning, human destruction and high-pressure electric shock, etc. CONCLUSION: In general, PMI can be correctly estimated by late postmortem phenomenon. When the cases included infection, poisoning and human destruction, we should estimate PMI with the comprehensive analysis.

Microfluidic preparation of a novel phoxim nanoemulsion pesticide against Spodoptera litura.

With continuous development of pesticide dosage forms, emulsifiable concentrates using large amounts of organic solvents are gradually obsoleted. Nanoemulsions with high water content have been developed and the preparation processes also evolved, but these processes still exist some problems, such as poor controllability and high energy consumption. Microfluidic is a controllable nanoemulsion preparation system which mainly applied to pharmaceutical synthesis. In this study, the pesticide phoxim nanoemulsion was prepared by microfluidic technology. The optimized formulation of phoxim nanoemulsion was composed of Tween 80 and pesticide emulsifier 500 as surfactant, hexyl acetate as oil, and n-propanol as co-surfactant. Moreover, when the flow rates of water and oil in the microfluidic system were adjusted to 5 µL/min and 20 µL/min, phoxim nanoemulsion was obtained with a cloud point/boiling point of 109 °C, a particle size of 21.5 ± 0.8 nm and a potential value of - 18.7 ± 0.6 mV. Furthermore, the nanoemulsion had a rapid release effect in vitro which could be fitted by the Ritger-Peppas model. The feeding toxicity of the phoxim nanoemulsion was higher than that of commercial formulation while the contact killing effect was higher than that of the active ingredient. Therefore, pesticide dosage was reduced and the insecticidal effect was enhanced by using phoxim nanoemulsions. These results also confirm the potential of microfluidics as a green process to produce pesticide nanoemulsions.